The TGF-β superfamily includes five distinct forms of TGF-β (Sporn and Roberts (1990) in Peptide Growth Factors and Their Receptors, Sporn and Roberts, eds., Springer-Verlag: Berlin pp. 419-472), as well as the differentiation factors vg-1 (Weeks and Melton (1987) Cell 51: 861-867), DPP-C polypeptide (Padgett et al. (1987) Nature 325: 81-84), the hormones activin and inhibin (Mason et al. (1985) Nature 318: 659-663; Mason et al. (1987) Growth Factors 1: 77-88), the Mullerian-inhibiting substance, MIS (Cate et al. (1986) Cell 45:685-698), osteogenic and morphogenic proteins OP-1 (PCT/US90/05903), OP-2 (PCT/US91/07654), OP-3 (PCT/WO94/10202), the BMPs, (see U.S. Pat. Nos. 4,877,864; 5,141,905; 5,013,649; 5,116,738; 5,108,922; 5,106,748; and 5,155,058), the developmentally regulated protein VGR-1 (Lyons et al. (1989) Proc. Natl. Acad. Sci. USA 86: 4554-4558), cartilage-derived growth factors CDMP-1, CDMP-2 and CDMP-3 (or GDF-5, GDF-6 and GDF-7), and the growth/differentiation factors GDF-1, GDF-3, GDF-9 and dorsalin-1 (McPherron et al. (1993) J. Biol. Chem. 268: 3444-3449; Basler et al. (1993) Cell 73: 687-702).
The proteins of the TGF-β superfamily are disulfide-linked homo- or heterodimers that are expressed as large precursor polypeptide chains containing a hydrophobic signal sequence, a long and relatively poorly conserved N-terminal pro region sequence of several hundred amino acids, a cleavage site, and a mature domain comprising an N-terminal region that varies among the family members and a more highly conserved C-terminal region. This C-terminal region, present in the processed mature proteins of all known family members, contains approximately 100 amino acids with a characteristic cysteine motif having a conserved six or seven cysteine skeleton. Although the position of the cleavage site between the mature and pro regions varies among the family members, the cysteine pattern of the C-terminus of all of the proteins is in the identical format, ending in the sequence Cys-X-Cys-X (Sporn and Roberts (1990), supra).
Recombinant TGF-β1 has been cloned (Derynck et al. (1985) Nature 316: 701-705), and expressed in Chinese hamster ovary cells (Gentry et al. (1987) Mol. Cell. Biol. 7: 3418-3427). Additionally, recombinant human TGF-β2 (deMartin et al. (1987) EMBO J. 6: 3673), as well as human and porcine TGF-β3 (Derynck et al. (1988) EMBO J. 7: 3737-3743; Dijke et al. (1988) Proc. Natl. Acad. Sci. USA 85: 4715), have been cloned. Expression levels of the mature TGF-β1 protein in COS cells have been increased by substituting cysteine residues located in the pro region of the TGF-β1 precursor with serine residues (Brunner et al. (1989) J. Biol. Chem. 264: 13660-13664).
A unifying feature of the biology of the proteins of the TGF-β superfamily is their ability to regulate developmental processes. These structurally related proteins have been identified as being involved in a variety of developmental events. For example, TGF-β and the polypeptides of the inhibin/activin group appear to play a role in the regulation of cell growth and differentiation. MIS causes regression of the Mullerian duct in development of the mammalian male embryo, and dpp, the gene product of the Drosophila decapentaplegic complex, is required for appropriate dorsal-ventral specification. Similarly, Vg-1 is involved in mesoderm induction in Xenopus, and Vgr-1 has been identified in a variety of developing murine tissues. Regarding bone formation, many of the proteins in the TGF-β supergene family, namely OP-1 and a subset of the BMPs, apparently play the major role. OP-1 (BMP-7) and other osteogenic proteins have been produced using recombinant techniques (U.S. Pat. No. 5,011,691 and PCT Application No. US 90/05903) and shown to be able to induce formation of true endochondral bone in vivo. BMP-2 has been recombinantly produced in monkey COS-1 cells and Chinese hamster ovary cells (Wang et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2220-2224).
Recently the family of proteins taught as having osteogenic activity as judged by the Sampath and Reddi bone formation assay have been shown to be morphogenic, i.e., capable of inducing the developmental cascade of tissue morphogenesis in a mature mammal (See PCT Application No. US 92/01968). In particular, these proteins are capable of inducing the proliferation of uncommitted progenitor cells, and inducing the differentiation of these stimulated progenitor cells in a tissue-specific manner under appropriate environmental conditions. In addition, the morphogens are capable of supporting the growth and maintenance of these differentiated cells. These morphogenic activities allow the proteins to initiate and maintain the developmental cascade of tissue morphogenesis in an appropriate, morphogenically permissive environment, stimulating stem cells to proliferate and differentiate in a tissue-specific manner, and inducing the progression of events that culminate in new tissue formation. These morphogenic activities also allow the proteins to induce the “redifferentiation” of cells previously stimulated to stray from their differentiation path. Under appropriate environmental conditions it is anticipated that these morphogens also may stimulate the “redifferentiation” of committed cells.
The osteogenic proteins generally are classified in the art as a subgroup of the TGF-β superfamily of growth factors (Hogan (1996), Genes & Development, 10:1580-1594), and are variously termed “osteogenic proteins”, “morphogenic proteins”, “morphogens”, “bone morphogenic proteins” or “BMPs” are identified by their ability to induce ectopic, endochondral bone morphogenesis. Members of the morphogen family of proteins include the mammalian osteogenic protein-1 (OP-1, also known as BMP-7, and the Drosophila homolog 60A), osteogenic protein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila homolog DPP), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and its murine homolog Vgr-1, BMP-9, BMP-10, BMP-11, BMP-12, GDF3 (also known as Vgr2), GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, BMP-13, BMP-14, BMP-15, GDF-5 (also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2 or BMP-13), GDF-7 (also known as CDMP-3 or BMP-12), the Xenopus homolog Vgl and NODAL, UNIVIN, SCREW, ADMP, and NEURAL.
Whether naturally-occurring or synthetically prepared, osteogenic proteins, can induce recruitment and/or stimulation of progenitor cells, thereby inducing their differentiation into chondrocytes and osteoblasts, and further inducing differentiation of intermediate cartilage, vascularization, bone formation, remodeling, and, finally, marrow differentiation. Furthermore, numerous practitioners have demonstrated the ability of these osteogenic proteins, when admixed with either naturally-sourced matrix materials such as collagen or synthetically-prepared polymeric matrix materials, to induce bone formation, including membraneous and endochondral bone formation, under conditions where true replacement bone would not otherwise occur. For example, when combined with a matrix material, these osteogenic proteins induce formation of new bone in large segmental bone defects, spinal fusions, clavarial defects, and fractures.
Bacterial and other prokaryotic expression systems are relied on in the art as preferred means for generating recombinant proteins. Prokaryotic systems such as E. coli are useful for producing commercial quantities of proteins, as well as for evaluating biological properties of naturally occurring or biosynthetic mutants and analogs. Typically, an over-expressed eukaryotic protein aggregates as an insoluble intracellular precipitate (“inclusion body”) in the prokaryote host cell. The aggregated protein is then collected from the inclusion bodies, solubilized using one or more standard denaturing agents, and then allowed, or induced, to refold into a functional state. Proper refolding to form a biologically active protein structure requires proper formation of any disulfide bonds.
Chemical synthesis may also be employed to produce protein constructs. Technology is widely available to permit routine, automated assembly of peptide chains. Techniques are known in the art which utilize enzymatic and chemical methods for coupling peptide fragments into synthetic protein molecules. See, e.g., Hilvert, Chem. Biol. (1994) 1(4): 201-03; Muir et al., Proc. Nat'l Acad. Sci. USA (1998) 95(12): 6705-10; Wallace, Curr. Opin. Biotechnol. (1995) 6(4): 403-10; Miranda et al., Proc. Nat'l Acad. Sci. USA (1999) 96(4): 1181-6; and Liu et al., Proc. Nat'l Acad. Sci. USA (1994) 91(14): 6584-8.
For example, the tertiary and quaternary structure of both TGF-β2 and OP-1 have been determined. Although TGF-β2 and OP-1 exhibit only about 35% amino acid identity in their respective amino acid sequences the tertiary and quaternary structures of both molecules are strikingly similar. Both TGF-β2 and OP-1 are dimeric in nature and have a unique folding pattern involving six of the seven C-terminal cysteine residues, as illustrated in FIG. 1A. FIG. 1A shows that in each subunit four cysteines bond to generate an eight residue ring, and two additional cysteine residues form a disulfide bond that passes through the ring to form a knot-like structure. With a numbering scheme beginning with the most N-terminal cysteine of the 7 conserved cysteine residues assigned number 1, the 2nd and 6th conserved cysteine residues bond to close one side of the eight residue ring while the 3rd and 7th cysteine residues close the other side. The 1st and 5th conserved cysteine residues bond through the center of the ring to form the core of the knot. The 4th conserved cysteine forms an interchain disulfide bond with the corresponding residue in the other subunit.
The TGF-β2 and OP-1 monomer subunits comprise three major structural elements and an N-terminal region. The structural elements are made up of regions of contiguous polypeptide chain that possess over 50% secondary structure of the following types: (1) loop, (2) α-helix and (3) β-sheet. Furthermore, in these regions the N-terminal and C-terminal strands are not more than 7 A° apart. The residues between the 1st and 2nd conserved cysteines (FIG. 1A) form a structural region characterized by an anti-parallel β-sheet finger, referred to herein as the finger 1 region (F1). A ribbon trace of the finger 1 peptide backbone is shown in FIG. 1B. Similarly the residues between the 5th and 6th conserved cysteines in FIG. 1A also form an anti-parallel β-sheet finger, referred to herein as the finger 2 region (F2). A ribbon trace of the finger 2 peptide backbone is shown in FIG. 1D. A β-sheet finger is a single amino acid chain, comprising a β-strand that folds back on itself by means of a β-turn or some larger loop so that the entering and exiting strands form one or more anti-parallel β-sheet structures. The third major structural region, involving the residues between the 3rd and 4th conserved cysteines in FIG. 1A, is characterized by a three turn α-helix referred to herein as the heel region (H). A ribbon trace of the heel peptide backbone is shown in FIG. 1C.
The organization of the monomer structure is similar to that of a left hand where the knot region is located at the position equivalent to the palm, finger 1 is equivalent to the index and middle fingers, the α-helix is equivalent to the heel of the hand, and finger 2 is equivalent to the ring and small fingers. The N-terminal region (not well defined in the published structures) is predicted to be located at a position roughly equivalent to the thumb.
In the dimeric forms of both TGF-β2 and OP-1, the subunits are oriented such that the heel region of one subunit contacts the finger regions of the other subunit with the knot regions of the connected subunits forming the core of the molecule. The 4th cysteine forms a disulfide bridge with its counterpart on the second chain thereby equivalently linking the chains at the center of the palms. The dimer thus formed is an ellipsoidal (cigar shaped) molecule when viewed from the top looking down the two-fold axis of symmetry between the subunits (FIG. 2A). Viewed from the side, the molecule resembles a bent “cigar” since the two subunits are oriented at a slight angle relative to each other (FIG. 2B).
However, not all solubilized heterologous proteins readily refold. Despite careful manipulation of refolding, the yields of properly folded, biologically active protein remain low. Many TBF-β family members, including BMPs, fall into the category of poor refolder proteins. While some members of the TBF-β protein family can be folded efficiently in vitro as, for example, when produced in E. coli or other prokaryotic hosts, many others, including BMP5, BMP6, and BMP7, cannot. See, e.g., EP 0433225, U.S. Pat. Nos. 5,399,677, 5,756,308 and 5,804,416.
A need remains for improved means for producing in vitro recombinant BMPs and other TGF-β family proteins using prokaryotic as well as eukaryotic host cells.